Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein–protein and protein–lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor–ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein-protein and protein-lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor-ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.

Workflow overview
The procedure starts with the separation of D. melanogaster heads for downstream enrichment of cellular membranes. Soluble proteins are separated from membrane proteins by means of centrifugation. Pelleted membrane proteins are solubilized in SMA 3:1 and incubated at room temperature to form the SMALPs. After the ultracentrifugation step, the supernatant contains a heterogeneous mixture of nanodiscs with a size between 5 and Published: Aug 05, 2023 15 nm that could be further examined using transmission electron microscopy. In order to target specific membrane proteins, an affinity purification is performed. In the example we give here, the α-BTX peptide is coupled to Sepharose beads and used to purify nAChRs. If necessary, the degree of enrichment of nAChRs is analyzed using western blot. In order to be able to identify the subunits of the receptor, the samples are digested using trypsin/lys-C mix and applied to LC-MS/MS. After analysis, a comparison against a non-enriched sample is performed to determine which protein subunits are specifically enriched.

Membrane protein enrichment and incorporation in SMALPs
The starting material can be adapted to the respective question. Material can range from human to bacterial cells, and from total tissues to whole organisms such as D. melanogaster (Gulati et  At this point, the heads can be stored at -80 °C for months before further extraction. 3. For cell lysis, add 1 mL of isotonic lysis buffer to approximately 0.8 g of separated heads. Mix the solution three times by vortexing and lyse the heads with 60 strokes in a 2 mL Dounce homogenizer with a pestle ( Figure  1A). 4. Perform membrane protein preparation by differential centrifugation-based fractionation, as described in Depner et al. (2014). This allows for a better separation of membrane proteins from soluble proteins. 5. Perform a pre-cellular clearance by a centrifugation step at 200× g for 5 min. This step allows the removal of undisrupted cells, which are not broken during the extraction process. 6. Centrifuge the supernatants by a series of differential centrifugation steps: 1,000× g for 5 min, 3,000× g for 10 min, 5,000× g for 10 min, and 9,000× g for 15 min. Perform all steps in a 4 °C refrigerated centrifuge. After each spin, transfer the supernatant into a new centrifugation tube and use the membrane pellets for western blot analysis. By employing this differential centrifugation-based fractionation strategy, plasma membranes are partially separated from other endomembranes, increasing sensitivity and specificity of the system. With higher and higher spins, the fractions that do not contain the plasma membrane are obtained. 7. Western blot analysis reveals which fraction is enriched for the plasma membrane and thus should be used in downstream processing ( Figure 1B). 8. Use fractions enriched in plasma membranes for the SMALP extraction. Resuspend membrane fractions (24-177 mg of wet pellet weight) in approximately 20-300 μL of 5% SMALP solution. 9. For efficient incorporation and formation of SMALPs, incubate fractions containing plasma membrane with 5% SMALP solution for 2 h at room temperature on a rocking platform. Finally, centrifuge at 100,000× g for 60 min at 4 °C and use the supernatant, which contains the SMALPs, for downstream analysis. SMALPs can be incubated on ice without precipitation, but low or rapid freezing can cause the nanodiscs to degrade.

Immunoblotting
Western blot analysis is used to investigate the enrichment of the plasma membrane proteins in fractions resulting from differential centrifugation. An anti-ATPase alpha 1 antibody is used to perform a western blot, acting as a plasma membrane marker ( Figure 1B), or it can be used to determine the degree of enrichment of nAChRs.
1. After the centrifugation steps, load fractions on a 4%-15% SDS-PAGE and transfer onto a 0.2 μm nitrocellulose membrane. 2. Use 5% skimmed milk powder dissolved in TBS-T for blocking and incubate membranes for 16 h at 4 °C with the anti-ATPase alpha 1 antibody (1:1,000 concentrated in blocking solution) followed by anti-mouse ECL  Figure 1C). This strain serves as a positive control for the successful enrichment of nAChRs using α-BTX affinity beads. Use 5% skimmed milk powder dissolved in TBS-T for blocking and incubate membranes for 16 h at 4 °C with the α-GFP antibody (1:1,000 concentrated in blocking solution) followed by anti-rat IgG antibody for 1 h. 4. Treat immunoblots with an ECL chemiluminescent detection solution exposed for 10 s to CL-XPosure films and visualize using an x-ray developer.

Coupling procedure of α-BTX to affinity beads
For this protocol, α-BTX-coupled beads are used to enrich for subunits of nAChRs. For other studies, alternative affinity enrichment strategies can be employed, such as specific antibodies used to target their corresponding membrane proteins. If ligands other than α-BTX are used, then ligand buffer solutions and coupling reactions must be optimized. Moreover, optimization of different linkers between affinity beads and ligands may be necessary if alternative ligands are employed. If antibodies or other ligands than α-BTX are used, the coupling and crosslinking reaction must be optimized. When using beads other than those described in this protocol, an optimization step should be performed.

Enrichment of nAChRs by α-BTX pull-down
If interacting molecules, such as toxin peptides other than α-BTX or antibodies, are used to enrich the desired membrane protein, the protocol should be adapted. The following protocol is developed with α-BTX, as this peptide has a high affinity for nAChRs (Dellisanti et al., 2007;Rahman et al., 2020). 1. Incubate SMALP discs (800-1000 μL of a 20-35 mg/mL protein extract, measured with a NanoDrop) with 200 μL of α-BTX-conjugated affinity beads for 16 h at 4 °C on a rotator. 2. Centrifuge the beads at 1,500× g for 5 min and wash two or three times, each for 10 min, with 1 mL of ice-cold TBS on a rotator at 4 °C. 3. Centrifuge the beads at 1,500× g for 5 min and selectively elute nAChRs twice with 100 μL of 1 M carbachol.
Perform these steps on a rotator at RT. 4. Centrifuge the beads at 1,500× g for 5 min. The supernatant should be transferred into a new clean tube. 5. Add ice-cold 100% acetone to the samples at a volume of four times the sample volume and mix by vortexing.
Leave proteins to precipitate for 16 h at -20 °C. An overnight precipitation using acetone allows the removal of contaminants including salts that may interfere with subsequent SDS-PAGE and analysis using western blotting. If further structural analysis of proteins is to be carried out using electron microscopy, this step may be skipped. 6. Centrifuge samples at 13,000× g for 15 min.

Sample preparation for liquid chromatography-mass spectrometry (LC-MS)
Gel pieces are excised from the Coomassie stained gel lanes; proteolytic digestion, performed using a commercial available Trypsin/Lys-C mix, is performed as described (Shevchenko et al., 2006;Saveliev et al., 2013). 1. Immerse the gel pieces in 50 mM NH4HCO3/50% ACN and shake with a V-32 Vortex Mixer at maximum speed for 10 min. Remove the supernatant and repeat these steps with 100% ACN; finally, dry in a speed vac for 20 min. 2. Reduce samples with 10 mM DTT in 50 mM NH4HCO3 at 56 °C for 1 h. Remove DTT completely to avoid any inhibition effects on IAA. 3. Carry out alkylation with 50 mM IAA in 50 mM NH4HCO3 at room temperature without light for 45 min.
Remove IAA. 4. Add 50 mM NH4HCO3 (fully cover the gel pieces); vortex for 5 min, centrifuge, and discard the solution. 5. Add 100% ACN to the gel pieces so they are completely covered with solution, shake for 10 min, and discard the solution. 6. Repeat these two steps (steps [4][5] and dry samples in a speed vac for 20 min. Add Trypsin/Lys-C buffer to the sample according to manufacturer's instructions and incubate for 45 min on ice. 7. Next, add 30 μL of 25 mM NH4HCO3 and incubate samples at 37 °C for 16 h. Cover the gel pieces with 20 mM NH4HCO3 and shake with a V-32 Vortex Mixer at maximum speed for 10 min. Collect the supernatant containing peptides. 8. Next, cover the gel pieces with 50% ACN/5% FA and shake for 20 min. Again, collect the supernatant containing the peptides. Repeat this step of 50% ACN/5% FA addition and shaking for 20 min and collect the supernatant containing the peptides. 9. Combine all the supernatants together and dry in a speed vac until completely dry. Store samples at -20 °C.

Peptide cleanup
Peptides are desalted using C-18 stage tips according to Rappsilber  Reload the flowthrough solution to make sure that as many peptides as possible bind to the C-18 material. For this, pipette again the peptide flowthrough solution onto the C-18 material and repeat the same centrifugation step. 8. Wash C-18 stage tips twice with 100 μL of 0.1% FA and centrifuge for 2 min at maximum speed. 9. Place the tips into a 1.5 mL low binding tube and elute peptides with 70% ACN 0.1% FA. 10. Centrifuge the C-18 stage tips at 2,000× g for 5 min to elute the peptides from the C-18 material. 11. Finally, dry peptides in a speed vac and store at -20 ° C before resuspending in 0.1% FA for further LC-MS/MS analysis.

Expected result
This protocol describes an approach to study the interaction of native membrane proteins with different ligands, like drug molecules. Membrane proteins are known to be difficult to extract in their native conformation, and therefore the development of a method that allows the successful enrichment of native membrane proteins for downstream analysis is of great importance. One of these downstream analyses could be, for example, a protein's interaction with different ligands and the better characterization of where these molecules bind to the membrane protein. Our protocol describes the enrichment by affinity beads of native nAChRs in SMALP preparations, and as expected, resulting subunits of these receptors can be identified by mass spectrometry. This helps to better characterize receptor-ligand interactions, and our protocol can be applied to various research questions, in a variety of different organisms.